DNA Segments Research Articles

OCCURRENCES OF TROPICAL ANGUILLIDS EELS IDENTIFIED THROUGH DNA BARCODES AT KEDURANG RIVER, SUMATRA ISLAND, INDONESIA

Understanding the geographic distribution of tropical anguillids is important for the conservation of these species. Delimitation of species distribution area is a fundamental task with important implications for the understanding of biodiversity and conservation. However, their geographic ranges are poorly understood, mostly because of the technical difficulties of identifying anguillids by morphological characteristics, especially at the glass eel stage. The current work aims to provide the information on recruitment of tropical anguilid eels in the Kedurang River by using analysis of species identification of an informative DNA segment of Cytochrome Oxidase Sub unit1. Results showed that PCR, sequencing and analysis of an informative DNA can be a useful complement to morphological study for more complete biodiversity assessments. The anguillid eels found in Kedurang River were identified and further validated as A. bicolor bicolor, A. interioris, A. bengalensis bengalensis, and A. marmorata through glass and adult eels. This study suggests the occurrence of A. interioris in Estuary of Sumatera River would provide the first confirmation for this species in the territory. The information can be useful for understanding the geographic distribution this species for the establishment and allocation of risk categories to species, both in national protection lists and in those of treaties and international conventions.

A strategy combining 3D-DNA Walker and CRISPR-Cas12a trans-cleavage activity applied to MXene based electrochemiluminescent sensor for SARS-CoV-2 RdRp gene detection

Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.

Construction of \xe0 la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins

BackgroundQconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process.ResultsWe report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro.ConclusionsWe refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Comparative genomic hybridization (CGH) in molecular diagnostics

Comparative genomic hybridization (CGH) is a powerful molecular cytogenetic approach for identifying chromosomal abnormalities. CGH allows researchers to scan whole genomes for changes in DNA copy numbers. Starting in 2004, the array CGH became an irreplaceable method for the detection of gene mutations in people with congenital and developmental abnormalities, such as intellectual disability, dysmorphic characteristics, developmental delay, or several congenital deformities without an obvious syndrome pattern. This review focuses on the evolution of array CGH technology and its use in molecular diagnostics and its advantages over older cytogenetic tools. This review further highlights special arrays developed in the past decade which detect small intragenic copy number changes as well as large DNA segments for the region of heterozygosity.

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair.

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.

Programming a "Crab Claw"-like DNA Nanomachine as a Super Signal Amplifier for Ultrasensitive Electrochemical Assay of Hg2.

Herein, with skillfully engaging stable T-Hg2+-T bonding, a "Crab Claw"-like DNA nanomachine with concise and highly efficient assembly and enhanced recognition/conversion efficiency was engineered as a super signal amplifier, which was united with Pd@Cu@Pt multimetallic mesoporous nanomaterials (Pd@Cu@Pt MMNs) for ultrasensitive electrochemical assay of mercury ions (Hg2+). Specifically, the formed "Crab Claw"-like DNA nanomachine could simultaneously trigger four same cascade DNAzyme cleavage reactions with the help of Mg2+ DNAzyme for markedly converting target Hg2+ to enormous DNA segments labeled with ferrocene (Fc), improving the detection sensitivity. Subsequently, the prepared Pd@Cu@Pt MMNs could not only show commendable electrochemical catalysis to Fc but also act as an excellent immobilization matrix for capturing and accumulating abundant Fc around them to further strengthen the electrochemical signal. As a result, the well-designed electrochemical sensor could achieve a low limit of detection of 3.58 fM in the range from 10 fM to 100 nM for Hg2+detection. This strategy offers a simple and rapid avenue to detect heavy metal ions and shows promising application potential for environmental pollutant monitoring.

A CpG island promoter drives the CXXC5 gene expression

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

Genes and Pseudogenes: Complexity of the RCCX Locus and Disease.

Copy Number Variations (CNVs) account for a large proportion of human genome and are a primary contributor to human phenotypic variation, in addition to being the molecular basis of a wide spectrum of disease. Multiallelic CNVs represent a considerable fraction of large CNVs and are strictly related to segmental duplications according to their prevalent duplicate alleles. RCCX CNV is a complex, multiallelic and tandem CNV located in the major histocompatibility complex (MHC) class III region. RCCX structure is typically defined by the copy number of a DNA segment containing a series of genes – the serine/threonine kinase 19 (STK19), the complement 4 (C4), the steroid 21-hydroxylase (CYP21), and the tenascin-X (TNX) – lie close to each other. In the Caucasian population, the most common RCCX haplotype (69%) consists of two segments containing the genes STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB, with a telomere-to-centromere orientation. Nonallelic homologous recombination (NAHR) plays a key role into the RCCX genetic diversity: unequal crossover facilitates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers. The results of these events increased the RCCX genetic diversity and are responsible of specific human diseases. This review provides an overview on RCCX complexity pointing out the molecular bases of Congenital Adrenal Hyperplasia (CAH) due to CYP21A2 deficiency, CAH-X Syndrome and disorders related to CNV of complement component C4.

Induction of a chromatin boundary in vivo upon insertion of a TAD border.

Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development.

CNVIntegrate: the first multi-ethnic database for identifying copy number variations associated with cancer.

Human copy number variations (CNVs) and copy number alterations (CNAs) are DNA segments (>1000 base pairs) of duplications or deletions with respect to the reference genome, potentially causing genomic imbalance leading to diseases such as cancer. CNVs further cause genetic diversity in healthy populations and are predominant drivers of gene/genome evolution. Initiatives have been taken by the research community to establish large-scale databases to comprehensively characterize CNVs in humans. Exome Aggregation Consortium (ExAC) is one such endeavor that catalogs CNVs, of nearly 60 000 healthy individuals across five demographic clusters. Furthermore, large projects such as the Catalogue of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE) combine CNA data from cancer-affected individuals and large panels of human cancer cell lines, respectively. However, we lack a structured and comprehensive CNV/CNA resource including both healthy individuals and cancer patients across large populations. CNVIntegrate is the first web-based system that hosts CNV and CNA data from both healthy populations and cancer patients, respectively, and concomitantly provides statistical comparisons between copy number frequencies of multiple ethnic populations. It further includes, for the first time, well-cataloged CNV and CNA data from Taiwanese healthy individuals and Taiwan Breast Cancer data, respectively, along with imported resources from ExAC, COSMIC and CCLE. CNVIntegrate offers a CNV/CNA-data hub for structured information retrieval for clinicians and scientists towards important drug discoveries and precision treatments. Database URL: http://cnvintegrate.cgm.ntu.edu.tw/

Portable Molecular Diagnostics Device for Identification of Asini Corii Colla by Loop-Mediated Isothermal Amplification

Asini Corii Colla (ACC; donkey-hide glue) is one of the most valuable tonic traditional Chinese medicines. Because of the large demand for gelatinous Chinese medicines, bovine or swine skin was sometimes used to make adulterated gelatine in recent decades. Food chemicals can greatly harm people’s health, and detecting chemicals in foods is extremely important. A loop-mediated isothermal amplification (LAMP) device with smartphone detection is demonstrated in this study for detecting the DNA of Asini Corii Colla. The complete system is composed of a hand-held box equipped with a smartphone, a cartridge heater, an ultraviolet LED, a disposable reaction tube, and a homemade thermal module. All the processes are powered by a set of rechargeable batteries. Comprehensive experiments of measuring temperature profiles are presented, which showed the accuracy of temperature under thermal control is less than 0.5 °C. By implementing one heating module with an ATmega328p-au microcontroller in the device, the DNA mixture is heated directly up to the reaction temperature within 5 min. Next, a DNA segment of Asini Corii Colla is utilized to evaluate the sensitivity of the DNA amplification in the portable device. A limit of detection to a concentration of 10−4 ng/μL is achieved. Real-time detection of Asini Corii Colla by a smartphone camera can be achieved using this portable device. The unique architecture utilized in this device is ideal for a low-cost DNA analysis system.

Ion-dependent DNA configuration in bacteriophage capsids

Bacteriophages densely pack their long double-stranded DNA genome inside a protein capsid. The conformation of the viral genome inside the capsid is consistent with a hexagonal liquid crystalline structure. Experiments have confirmed that the details of the hexagonal packing depend on the electrochemistry of the capsid and its environment. In this work, we propose a biophysical model that quantifies the relationship between DNA configurations inside bacteriophage capsids and the types and concentrations of ions present in a biological system. We introduce an expression for the free energy that combines the electrostatic energy with contributions from bending of individual segments of DNA and Lennard-Jones-type interactions between these segments. The equilibrium points of this energy solve a partial differential equation that defines the distributions of DNA and the ions inside the capsid. We develop a computational approach that allows us to simulate much larger systems than what is possible using the existing molecular-level methods. In particular, we are able to estimate bending and repulsion between the DNA segments as well as the full electrochemistry of the solution, both inside and outside of the capsid. The numerical results show good agreement with existing experiments and with molecular dynamics simulations for small capsids.

Two carbapenem-resistant ST1:ST231:KL1:OCL1 Acinetobacter baumannii strains recovered in Tehran, Iran, carry AbaR31 in the chromosome and AbaR4 and TnaphA6 in a RepAci6 plasmid.

ObjectivesTo analyse the context of genes conferring antibiotic resistance in two carbapenem-resistant Acinetobacter baumannii isolates recovered in Tehran, Iran.MethodsThe antibiotic resistance phenotype for 28 antibiotics was determined using disc diffusion. The whole genome sequences of ABH008 and ABS200 were determined using the Illumina HiSeq X Ten platform. Resistance genes were identified using ResFinder and multilocus sequence types were determined using the Oxford and Institut Pasteur schemes.ResultsIsolates ABH008 and ABS200, recovered in 2012 and 2013, respectively, in two different Tehran hospitals, belong to the common global clone 1 lineage, ST1IP and ST231OX. They are resistant to sulfamethoxazole, tetracycline, gentamicin, amikacin, third-generation cephalosporins and carbapenems. Despite being isolated in different hospitals, phylogenetic analysis indicated they are closely related. Consistent with this, both isolates carry catA1, sul1, aacC1 and aadA1 in a novel variant of the AbaR3-type resistance island, named AbaR31. Both isolates are resistant to amikacin and carbapenems owing to aphA6 and oxa23, respectively. The oxa23 gene is located in the AbaR4 resistance island, and aphA6 in TnaphA6, and both mobile elements are in an ∼90 kbp plasmid encoding the putative RepAci6 replication initiation protein. Resistance to third-generation cephalosporins is due to the acquisition by homologous recombination of a 5 kb DNA segment that contains ISAba1-ampC from a ST623 strain.ConclusionsThe resistance gene complements of ABH008 and ABS200 were found in AbaR31 and a plasmid that encodes RepAci6. The close genetic relationship of ABH008 and ABS200, despite each being recovered from different hospitals, indicates transmission between the two hospitals.

The BRCT Domain of PARP1 Binds Intact DNA and Mediates Intrastrand Transfer

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in the response to DNA damage and is the target of four different clinical inhibitors used for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation via destabilization of a subdomain of the catalytic domain, and activated PARP1 utilizes NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. Here we show that intact DNA makes contacts with the BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy (cryoEM) and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the ‘monkey-bar mechanism’ that mediates transfer of PARP1 from one DNA segment to another.

OTEH-12. Assessing Adaptive Responses to Loss of Extrachromosomal DNA Amplification

BackgroundOncogene activation through somatic gene amplification happens frequently in GBM, with over 70% of these tumors presenting amplification of at least one putative driver gene, oftentimes in small extrachromosomal circular DNA segments composed of chromatin (ecDNA). A molecularly diverse and representative panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX), which retain the original genomic abnormalities and ecDNA amplifications, was employed to assess adaptive response to the absence of ecDNA amplification.MethodsWe have isolated ecDNA negative cell populations from two patient-derived models. HF3035 harbors a MET amplification and HF3253 harbors a PDGFRA constitutively active genomic rearrangement and extrachromosomal amplification. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicate PDXs and 4 clones from 4 in vitro technical replicates) and 12 HF3035 population (ecDNA+/-: 6 clones from 3 biological replicate PDXs).ResultsNonparametric differentially expressed gene (DEG) analysis using NOISeqBio (R/Bioconductor), identified 564 differentially expressed genes (482 upregulated in ecDNA(-)) employing a stringent false discovery rate of 0.05. Genes significantly associated with PDGF stimulation, central carbon metabolism, and H3K27me3 were downregulated in ecDNA(-), while genes significantly associated with astrocytic processes, neuronal differentiation, and EGFR signaling were upregulated in ecDNA(-) (EnrichR). We employed an additive linear model with PDX serving as a blocking factor to compare ecDNA+ and ecDNA- populations in both models (R/edgeR). 2071 genes were upregulated in ecDNA+ PDX specimens and 2365 genes were downregulated. Specifically, E2F targets were highly enriched in ecDNA+ populations, in addition to mRNA pre-processing. ecDNA loss primarily targeted glycogen metabolism, NTRK signaling, and inositol phosphate catabolism.ConclusionsWe have identified PDX-specific and non-specific features to an adaptive response to the loss of ecDNA amplification. Notably, a signature adaptation is an upregulation of seemingly redundant receptor tyrosine kinases.

Analysis of DNA by Southern Blotting.

Southern transfer and hybridization are used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA for which specific probes are available. Genomic DNA is first digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a standard agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually a nylon or nitrocellulose membrane). The DNA attached to the membrane is hybridized to a labeled DNA, RNA, or oligonucleotide probe, and bands complementary to the probe are located by an appropriate detection system (e.g., by autoradiography). By estimating the size and number of the bands generated after digestion of the genomic DNA with different restriction enzymes, singly or in combination, it is possible to place the target DNA within a context of restriction sites.

Abstract 2096: Genome wide remodeling of DNA methylation and CpG hypermethylation is tightly linked to acquired resistance in testicular cancer

Abstract Acquired resistance to chemotherapy is perhaps the major barrier to cure of advanced cancers. Testicular germ cell tumors (TGCTs) are the most common cancers of young males. TGCTs are one of the few solid tumors that are cured at a high rate with standard cisplatin-based chemotherapy. However, TGCTs become resistance to cisplatin resulting in poor outcomes. While the mechanisms accounting for cisplatin hypersensitivity in TGCT remain elusive, it has been suggested that DNA hypermethylation may play a role. However, there is little direct experimental evidence to support this claim. In the present study, we have leveraged unique isogenic cell models to provide strong evidence for net DNA hypermethylation as a key mechanism leading to acquired cisplatin resistance in TGCT cells. Genome-wide DNA methylation analysis revealed that hypomethylated CpG loci were enriched for SUZ12 and EZH2 binding and H3K27me3 sites. In contrast hypermethylated CpG loci were significantly enriched for CTCF and RAD21 domains as well as repressive DNA segments indicating the global remodeling of nuclear chromatin structure in resistant cells. Integrative computational analysis of RNA-seq and EPIC DNA methylation array data revealed a strong inverse correlation between gene expression and CpG gene promoter methylation. This analysis also revealed a strong bidirectional shift between gene promoter and gene body DNA methylation that correlated closely with upregulated expression of PRC2 target genes and down regulation of tumor suppression genes in resistant cells. Hence the data further suggests a functional interplay between DNA methylation and the PRC2 pathway that modulates cisplatin sensitivity of TGCTs. Further studies including H3K27me3 ChIP-seq are ongoing to further elucidate this relationship. In conclusion, our finding supports the hypothesis that genome wide remodeling of DNA methylation is associated with cisplatin hypersensitivity and resistance of TGCTs and further supports the use of hypomethylation therapy for relapsed TGCT patients. Citation Format: Zeeshan Fazal, Ratnakar Singh, Megan Tomlin, Doha Shokry, Aleyah Hattab, Sarah Spinella, Michael Spinella. Genome wide remodeling of DNA methylation and CpG hypermethylation is tightly linked to acquired resistance in testicular cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2096.

Putative genes for resistance to gray leaf spot of maize based on genomic resequencing using recombinant inbred lines

AbstractGray leaf spot (GLS) of maize (Zea mays L.), caused by Cercospora spp., inflicts serious yield losses. Four recombinant inbred lines (RILs) were developed from the [(YML32×Q11) × Q11] backcross population. Two flanking markers, GZ204 and IDP5, defining quantitative trait locus (QTL) qRgls1 for resistance to GLS, were detected in three RILs (RL1_1, RL1_2, and RL2_1) but not in RL2_2. This QTL is located on chromosome 8 in bin 8.02. The field resistance of RL2_2 was, however, similar to that of RL1_1 and RL1_2. The RL2_2 was found to possess a unique DNA segment (Zm00001d008467) that was not found in the other three RILs. We tested the hypothesis that Zm00001d008467 is a candidate gene associated with resistance to GLS. Sequencing showed that clean read counts for RL1_1, RL1_2, RL2_1, and RL2_2 were different and that single‐nucleotide polymorphisms (SNPs)/Exonic SNPs in RL1_1, RL1_2, RL2_1, and RL2_2 were 4908618/14042, 2423243/10668, 4512193/12577, and 2321974/6731, respectively. Results revealed that one of the three exclusive extron nonsynonymous SNPs (Zm00001d008467_T003) had caused a change in a protein that is a wall‐associated non–arginine‐aspartate (non‐RD) receptor‐like kinase. This protein is suspected to trigger resistance to GLS. Our results confirm that Zm00001d008467 is a candidate gene, located in chromosome 8 in bin 8.02, that is associated with GLS resistance. We concluded that the exclusive extron nonsynonymous SNPs in the QTL that had changed the structure and function of the coding protein was responsible for the GLS resistance in RIL2_2 in the absence of resistance QTL qRgls1.

Conformational Dynamics of Histone H3 Tails in Chromatin.

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.